DESCRIPTION: The long-term research goal of this project is to understand in molecular terms how intracellular signal transduction is mediated by protein phosphorylation and dephosphorylation. This proposal intends to study the roles of protein phosphatases in MAPK (Mitogen-Activated Protein Kinase) cascades, using budding yeast (Saccharomyces cerevisiae) and cultured human cells. The regulatory mechanism of a MAPK cascade is important to mammalian oncogenesis, because many oncogenes (e.g., Src, Ras, Raf, and Mos) exert their effects through hyper-activation of MAPKS. MAPK cascades are regulated by dephosphorylation as well as by phosphorylation. In spite of intensive and extensive research efforts, however, much remains to be learned about phosphatases that down-regulate MAPK cascades. This project will focus on two specific types of protein phosphatases: protein tyrosine phosphatase (PTPase) and protein serine/threonine phosphatase type 2C (PP2C). The yeast HOG (High Osmolarity Glycerol response) signaling pathway will be studied as a model, because two PTPases (PTP2 and PTP3) and a PP2C (PTC1) have been implicated in the HOG MAPK cascade. Yeast is an ideal model system to study the general principles governing intracellular signal transduction, not only because it is genetically tractable, but also because its signaling pathways have many features in common with human cells. The specific aims of this proposal are: 1) Study on the regulatory mechanism of the PTP2 and PTP3 tyrosine phosphatase activities by the HOG1 MAP Kinase. The post-translational activation of PTP2/PTP3 will be examined using in vitro assay systems. Whether the PTP2/PTP3 activities are regulated by protein phosphorylation will be tested. Also, the regulatory mechanism of the PTP2/PTP3 activities will be studied using genetic methods. 2) Study on the role of PTC1 and other type 2C protein phosphatases in yeast MAPK signal transduction. The physiological substrate of PTC1 will be defined using genetic epistasis test and an in vitro assay system. The role o the other type 2C protein phosphatases in yeast will be also examined. 3) Study on the role of the mammalian type 2C protein phosphatases in MAPK signal transduction. The regulatory roles of PP2Ca and PP2Cb on mammalian MAPKs will be studied both in vivo and in vitro.